Our pursuit of the mechanism of hormonal regulation of gluconeogenesis in isolated hepatocytes will focus on pyruvate kinase and possibly fructose dephosphatase as well. Specifically we will investigate: 1) the control by hormones of flux through pyruvate kinase in intact hepatocytes, 2) the quantitative significance of recycling of phosphoenolpyruvate to pyruvate with physiological substrates such as lactate and alanine, 3) the role of ions, particularly Ca2 ion, on activity of and flux through pyruvate kinase. The effect of Ca2 ion on purified pyruvate kinase will also be studied, 4) Whether or not control of pyruvate kinase activity by hormones is mediated by a change in the phosphorylation state of the enzyme and whether this is a relevant control mechanism in vivo, 5) Whether or not fructose diphosphatase activity is also under hormonal control, since glucagon lowers FDP levels in hepatocytes, 6) Whether fructose diphosphatase is phosphorylated in vitro and/or in vivo and whether the phosphorylation is controlled by hormones, 7) The relevance of hormone induced ion flux to flux through fructose diphosphatase. BIBLIOGRAPHIC REFERENCES: Pilkis, S.J., Riou, J.P., and Claus, T.H., Hormonal control of dihydroxyacetone and glycerol gluconeogenesis in isolated hepatocytes. J. Biol. Chem. (1976) in press. Pilkis, S.J., Claus, T.H., Riou, J.P., and Park, C.R., Possible role of pyruvate kinase in the control of dihydroxyacetone gluconeogenesis. Metabolism, in press, Nov. 1976.